Mus musculus breed:R26-Cas9 Knock In
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PROVIDER: PRJNA739880 | ENA |
REPOSITORIES: ENA
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Project description:Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway
| PRJNA742124 | ENA
Project description:The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation, but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).
2021-06-29 | GSE179089 | GEO
Project description:Galectin-3 is a carbohydrate binding protein implicated in cell-cell interaction and cell growth. Interestingly, recent studies suggest that galectin-3 is involved in cell metabolism and linked to diabetes and cancer. How galectin-3 might act as a diabetogenic and tumorogenic factor remains to be investigated. Here we report that intracellular galectin-3 is physically associated with Rag GTPases and Ragulator of the mTORC1 signaling machinery on lysosomes. We show that galectin-3 senses lipopolysaccharide (LPS) to facilitate the interaction of Rag GTPases and Ragulator, leading to activation of Rag GTPases thus lysosomal loading of mTOR and activation of mTORC1. Integrative genomics and transcriptomics analyses reveal that the LPS/galectin-3-Rag GTPases/Ragulator-mTORC1 axis regulates a cohort of genes including GLUT1 (glucose transporter member 1) that functions in glucose uptake and glycolysis and HK2 (hexokinase 2) and PKM2 (pyruvate kinase M2) that catalyze the first and last step of glycolysis, respectively. Indeed, galectin-3 deficiency severely compromises LPS-promoted glycolysis. Importantly, in diabetes patients, the expression of HK2 was significantly reduced, and in hepatocellular carcinoma (HCC), thyroid cancer, and prostate cancer, galectin-3 is highly expressed, and its level of expression is positively correlated with that of HK2 and PKM2 and negatively correlated with the prognosis of HCC patients. Our study unravels that galectin-3 is a sensor of LPS, an important modulator of the mTORC1 signaling, and a critical regulator of glucose metabolism, adding to the understanding of diabetogenesis and tumorigenesis.
2022-10-25 | GSE211784 | GEO
Project description:Mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is activated by nutrition sufficiency signals and extracellular growth signals. mTORC1 acts the hub that integrates these inputs to orchestrate number of cellular responses such as translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, the scaffold protein which specifically regulates any single downstream signaling molecule has not been identified to date. Here we show the heteropentamer protein complex Ragulator is critically required to regulate nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacks Ragulator but maintained total mTORC1 activity. The clone showed a markedly enhanced nuclear translocation of TFEB even in nutrition-sufficient state, despite the full mTORC1 activity. As a cellular phenotype, the number of lysosomes were increased by 10 times in the Ragulator-deficient clone. These findings suggest that mTORC1 essentially requires the scaffold Ragulator to regulate the subcellular location of TFEB. Our finding implicates that mTORC1 has other scaffold proteins that regulate downstream molecules specifically.
2021-08-08 | GSE171357 | GEO
Project description:Differentiation is critical for epithelial cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, lysosomal cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of Rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in a zebrafish model of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 signaling and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.
2025-05-13 | PXD040450 | Pride
Project description:This submission is associated with a paper by Hesketh et al. that identifies the GATOR-Rag GTPase pathway as a negative regulator of mTORC1 activation by lysosome-derived amino acids.
2019-07-30 | MSV000084159 | MassIVE
Project description:Gasdermin D (GSDMD) is the executioner of pyroptosis, which is important for host defense against pathogen infection. After activation, caspase-mediated cleavage of GSDMD liberates an N-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and subsequent release of proinflammatory cytokines. How this process is spatiotemporally controlled to promote pyroptosis in cells has been a fundamental, unaddressed question. Here, we identify GSDMD as a substrate for reversible S-palmitoylation on cysteine 192 (Cys192) in response to lipopolysaccharide (LPS) stimulation. We found that the palmitoyl acyltransferase DHHC7palmitoylates GSDMD to direct its cleavage by caspases in pyroptosis by promoting the interaction of GSDMD and caspases. We further show that after GSDMD cleavage, palmitoylation of GSDMD-NTpromotes its plasma membrane translocation and binding, and then acyl protein thioesterase 2 (APT2) depalmitoylates GSDMD-NT to unmask the Cys192 residue to promote oxidation-mediated oligomerization and pyroptosis. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, extends the survival of mice from LPS-induced lethal septic shock and sensitizes mice to bacterial infection. Thus. our findings reveal a model through which a palmitoylation-depalmitoylationrelay spatially and temporally controls GSDMD activation in pyroptosis.
2024-02-25 | GSE252911 | GEO
Project description:Gasdermin D (GSDMD) is the executioner of pyroptosis, which is important for host defense against pathogen infection. After activation, caspase-mediated cleavage of GSDMD liberates an N-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and subsequent release of proinflammatory cytokines. How this process is spatiotemporally controlled to promote pyroptosis in cells has been a fundamental, unaddressed question. Here, we identify GSDMD as a substrate for reversible S-palmitoylation on cysteine 192 (Cys192) in response to lipopolysaccharide (LPS) stimulation. We found that the palmitoyl acyltransferase DHHC7palmitoylates GSDMD to direct its cleavage by caspases in pyroptosis by promoting the interaction of GSDMD and caspases. We further show that after GSDMD cleavage, palmitoylation of GSDMD-NTpromotes its plasma membrane translocation and binding, and then acyl protein thioesterase 2 (APT2) depalmitoylates GSDMD-NT to unmask the Cys192 residue to promote oxidation-mediated oligomerization and pyroptosis. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, extends the survival of mice from LPS-induced lethal septic shock and sensitizes mice to bacterial infection. Thus. our findings reveal a model through which a palmitoylation-depalmitoylationrelay spatially and temporally controls GSDMD activation in pyroptosis.
2024-02-25 | GSE252893 | GEO
Project description:Differentiation is critical for epithelial cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, lysosomal cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of Rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in a zebrafish model of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 signaling and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.
2023-03-08 | GSE226397 | GEO
Project description:Differentiation is critical for epithelial cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, lysosomal cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of Rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in a zebrafish model of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 signaling and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.
2023-03-08 | GSE226396 | GEO